ABSTRACT The calvaria (upper part of the skull) comprises plates of bone and fibrous joints (sutures and fontanels), and the balance between the two components is crucial. Craniosynostosis (premature loss of suture(s)) occurs at a frequency of 1/2000 births, and it leads to a dysmorphic skull that can further affect brain and orofacial development. Current treatment of craniosynostosis often involves invasive surgeries at young ages, with risks for significant morbidity and even mortality. Therefore, improving the methods of intervention for this defect is of great importance to public health. The long-term goal of our research is to obtain comprehensive understanding of the molecular genetic regulation of calvarial development, which can lead to innovative strategies to treat and prevent related birth defects. During embryonic development, the head mesenchyme primordium of the calvaria completely encases the brain from early stages. Subsequently, the calvarial bone starts to develop from the mesenchyme on the lateral sides of the brain just above the eye (`supra-orbital' mesenchyme, SOM), and expands gradually toward the vertex. In contrast, the mesenchyme positioned at the vertex from the beginning (`early migrating' mesenchyme, EMM) does not initiate ossficiation, and contributes only to the soft tissue such as the sutures and the dermis. This spatial restriction in bone formation is crucial to making the properly patterned calvaria because it allows the vertex to be occupied by sutures and fontanels instead of bone. To date, little is known about the factors that underlie this regional difference in the developmental program within the head mesenchyme. Based on our preliminary data, we hypothesize that EMM is intrinsically programmed to resist osteogenic induction, and that LMX1B (LIM homeobox transcription factor 1b) is a key anti-osteogenic factor in this context. Therefore, the goal of this proposal is to elucidate the molecular mechanism controlling the osteogenic competence of EMM with a focus on LMX1B. We will define the spatial and temporal specificity of LMX1B function during calvarial development, and investigate the effect of LMX1B on the function of osteogenic signals. Furthermore, we will use genome-wide approaches to define the genetic programs specific to EMM and SOM, and identify Lmx1b-downstream genetic network that regulates this patterning of the head mesenchyme. The outcome of our research will provide crucial insights into the regulation of early stages of calvarial development and identify novel mechanisms and players that can contribute to the pathogenesis of craniosynostosis.